We have refined the first in vitro bioassay system for qualitative and quantitative analysis of the testis stimulating factor. Before, the only method for determining the presence of the testis stimulating factor utilized testicular artery and vein catheterization of intact cynomolgus monkeys. Using this bioassay, we were able to show that this factor acts as a protein in ammonium sulfate precipitation. Using size exclusion chromatography. we can show two peaks of bioactivity which are distinct from LH. We began a collaboration with Dr. David Klapper at the University of North Carolina to help us isolate this factor. We have used the standard isolation techniques of size, ion exchange, and immunoaffinity chromatography. We have further studied the second messenger system used by this factor as it may have unique clinical significance because it is able to stimulate virilization as well as spermatogenesis in the absence of gonadotropins. It may further be used as a model of a protein that can significantly affect reproduction and may further elucidate other pathways for reproductive effects of toxins.